Artemia with Synbiotics Enrichment Improves Resistance Against Vibrio parahaemolyticus AHPND of Litopenaeus vannamei Larvae

Shrimp, a high-protein food commodity, is one of the world's fastest-growing food-producing sectors. The present research aimed to find out the survival and growth of Litopenaues vannamei Post Larva (PL) and the resistance against VpAHND and stress salinity. 1500 PL were reared to two sets of experiments at the density of 50 PL. L -1 . The first set is purposed to determine the PL growth, resistance to Vibrio paramaemolyticus AHPND challenge and stress salinity. The second set is purposed to determine the survival rate. A Completely randomized design (CRD) with five treatments and three replications was conducted. The treatments are Artemia enrichment with different Alginate doses and probiotics (400, 600, 800 ppm Alg+pro), probiotics (Pro), and control without any synbiotics addition. PL was reared in 14 days. The survival rate, and weight gain were calculated. At the end of the experiment, 10 PL was challenged against VpAHPND at 1 x 10 7 CFU mL -1 by immersion methods. Twenty PL was exposed to stress salinity and shocked from 25 ppt to 0 ppt. The best survival rate ((78±2%), and tolerance to osmotic stress was reached at PL fed on a combination of alginate and FNCC-002 Lactobacillus bulgaricus probiotics (p<0.05). PL fed on Artemia enriched probiotics reached the highest resistance to severe VpAHNPD. The weight gain among treatments is similar. It can be concluded that synbiotics of alginate as prebiotics and FNCC-004 probiotics work synergically and this might be interrelated with immune response.


INTRODUCTION
Litopenaeus vannamei is one of the main part aquatic products among fishery trading commodities globally, and the most important species of shrimp culture in Indonesia.The fact of reducing wild-capture harvest i.e. 3.1 million tonnes of L. vannamei farming reported nearly 6.5 million tonnes (56%) from the global shrimp production in 2019 (FAO, 2021).
In practice, one of the most important biosecurity aspects for enhancing early culture stages including post larva and providing an effective shrimp farming grow-out phase is to find out high-quality rearing of post larva (PL) shrimp throughout the nursery phase (Rodríguez-Olague et al., 2021).The success of nursery phase depends on rigorous organize to adequate feeding, water quality, and other specialized management (García-Guerrero et al., 2015).Particularly, the shrimp PL stage needs a highly nutritious diet throughout nursery rearing that occupies high quality marine ingredients (Ayisi et al., 2017).Artemia nauplii is a vital crustacean larval live food that can be cultured in huge numbers at large densities (Sorgeloos, 1980).However, Artemia nauplii cannot meet up the nutritional supplies for larval development when used as the singular food.Several researchers has been reported Artemia enrichment by carotenoid-rich microalgae (Gui et el., 2022), Dunaliella salina (Bhuvaneshwari et al., 2018) and Isochrysis galbana (Martelli et al., 2020) which delivered the basic nutrients for the growth and development of nauplii.
The health status of shrimp and aquatic animals is closely interrelated to gut microbiota and the environment (Lima et al., 2021).Valuing the effects of feeding trials on the performance of PL shrimp not only assesses their growth and survival but also their quality through V. parahaemolyticus AHPND challenge and a stress test (Yudiati et al., 2020).
Alginate, the water extracted from Sargassum sp cell wall is well known as immunostimulant agent (Isnansetyo et al., 2015;Yudiati et al., 2016;Yudiati et al., 2019) and worked synergically as synbiotics with Lactobacillus bulgaricus probiotics (Yudiati et al., 2021).Furthermore, Du et al. (2019) denoted that Lactobacillus pentosus is able to increase disease resistance, gut bacterial diversity, and growth performance.We have evaluated that sodium alginate has a good potency as synbiotics and had a synergetic mechanism with L. bugaricus probiotics to counteract the combat of three single and combined Vibrio spp. in Artemia as biomodel (Yudiati et al., 2021).The key to this mechanism is the enhance of the immune system and the balance of microbiota.Gut microbiota could provide a blockade against pathogen invasion, start promoting host nutrient absorption through extracellular product secretion, and accelerate immune response (Libertucci & Young, 2019).
Salinity variation and fluctuation, especially in tropical areas is risky, due to rain and precipitation.Among the stress test methods, the assessment of resistance to salinity shock is suitable and effective tool for evaluating the quality of penaeid PL, particularly in tropical area such as Indonesia.
There is still lack of current information concerning the performance L. vannamei PL fed on Artemia enriched with combination of sodium alginate and probiotics which then followed by challenged against high virulence V. parahaemolyticus AHPND as well as stress salinity.This study is aimed to find out the survival and growth of L vannamei PL and the resistence against VpAHND and stress salinity.

Sargassum sp
This research was done from July to September 2022.The extraction and production of sodium alginate were basically based on Yudiati et al., (2016).Four grams of dried Sargassum sp. was added to 100 mL aquadest, 5 g Na2CO3, dan 1.86 g EDTA and mixed.HCl was added to manage the desirable pH, stirred and filtered.The KCl was then put into the filtrate and followed by absolute ethanol for precipitation.Centrifugation was then administered for pellet production and dried up.Our sodium alginate was fit to the standard alginate (Sigma®, USA) (Yudiati et al., 2016), with 217.5 KDa molecular weight at 89,95% acetylation degree (Yudiati et al., 2018).

Preparation of L. bulgaricus FNCC-004 Probiotics and sodium alginate
FNCC-004 was provided by Tropical Marine Biotechnology Lab., Dept. of Marine Science, Diponegoro University.Prior to these, all glassware, media and materials were sterilized using an autoclave and spraying with 70% alcohol inside Laminar Air Flow under UV exposure (Guridi et al., 2019).
One ose of FNCC-004 cultured in de Man, Rogosa dan Shape Agar/MRS Agar media was suspended to 100 mL Nutrient Broth/NB (Merck, USA) and 5% of MRS broth media dan incubated for 24 hrs at 37 o C (Yudiati et al., 2020).The synbiotics were prepared by mixing the FNCC-004 and sodium alginate.A serial concentration of alginate 0, 400, 600, dan 800 ppm was adjusted by adding 0.006, 0.012, and 0.018 g Alginate and diluted into 20 mL liquid culture of FNCC-004 dan 180 mL sterile sea water.The FNCC-004 and alginate fermentation was done by stirring at 150 rpm at room temperature (Yudiati et al., 2021).

Nauplii Artemia Enrichment
One gram of Artemia cyst (Supreme Plus®, Golden West Artemia) was weighted and hatched in strong aerated 1,000 mL sterile seawater.Along 16-18 hrs nauplii was hatched, collected, and ready to use (Rudtanatip et al., 2019).The enrichment of Artemia nauplli was done by immersion.250-500 nauplii Artemia was immersed in 50 mL fermented probiotics and alginate for one hour (Yudiati et al., 2021).The enriched Artemia is ready to use as natural feed for L. vannamei PL.

Experimental Design and L. vannamei Post Larva Feeding Trial
This is an experimental laboratory research.A Completely randomized design (CRD) with five treatments and three replications was conducted.The treatments are Artemia enrichment with different Alginate dose and probiotics (400, 600, 800 ppm Alg+pro), probiotics (Pro), and control without any synbiotics addition We have done the experiment in two sets.First set to determine growth performance, bacterial challenged and stress salinity, and another set to determine survival rate.1,500 L. vannamei PL (0.01 mg) were acclimated in 45 L container with 30 L in volume and was adapted with 25 ppt seawater for 24 hrs.
Firstly, one set trial of growth performance, bacterial challenged and stress salinity was prepared.In next day, 750 PL were selected and stocked to round bottom flask (1,000 mL in volume) with 25 ppt seawater media and started feeding on the enrichment Artemia according to the treatments.The density of each flask was 50 PL.The rearing period is 14 days and the feeding frequency was five times/day (07.00, 10.00, 13.00, 16.00, dan 19.00).To maintain the water quality, the shrimp faecal was siphoned every two days.The water exchanged was 30% from the total volume.At 14 days of rearing, 5 PL were picked up randomly and observed under microscope to assess the PL weight.Other 10 PL was challenged against Vibrio parahaemolyticus AHPND and 20 PL for salinity stress with 0 ppt salinity exposure.Another similar set of experiment was applied to determine the PL survival rate.The survival rate was counted at the end of experiment (14 days).

Preparation of Vibrio parahaemolyticus AHPND and Challenge Test
V. parahaemolyticus (VpAHPND) strain was purchased from Main Center of Brackishwater Aquaculture, Jepara.VpAHPND was recultured in laboratory and stocked Alkaline Peptone Water (APW, Merck), and followed by culturing in liquid APW and left for 24 hrs to grow, then followed by centrifugation at 4,200 rpm, for 15 minutes.Sterile seawater was used for pelleted bacterial cell dilution.In terms of bacterial challenged to PL, the density of VpAHPND was evaluated and determined in spectrophotometer at 600 nm.This 2.0 optical density is equal to 1 x 10 9 CFU mL -1 (Kongchum et al., 2022).
The challenge methods was modified from Balcázar et al. (2007).Ten PL was separated to the new rounded bottom flask and set up according to the treatments.Flasks were previously filled with 99 mL sterile seawater.One mL VpAHPND (1 x 10 7 CFU mL -1 ) were introduced to the flasks.The survival rate was monitored every day until 7 days.

Stress Salinity Exposure Test
The stress salinity was modified from Richardson et al. (2021).At the end of experiment, 20 survived PL from 25 ppt seawater media was taken and moved to petridish (50 mL freshwater, 0 ppt) according to the treatments.The mortality of PL was monitored and counted every 10 minutes for 60 minutes exposure.

Data Analysis
The data was statistically analyzed using R-Studio to determine the differences between treatments.The data was confirmed by one-way analysis of variance (ANOVA) to determine whether the treatment had a significant effect (p<0.05).To determine the differences, LSD was applied for the survival rate, weight gain, VpAHPND challenge and stress salinity exposure test data.

RESULTS AND DISCUSSION
Results shows that L. vannamei PL fed on Artemia enriched with alginate and probiotics reach the best survival rate at (78±2%), follow with other treatments and control reach the lowest PL survival rate at (43±14.75%).(Figure 1).
Sodium alginate which extracted from Sargassum sp.cell wall is a polysaccharide and constructed with mannuronic and guluronic monosaccharides (Dragdet & Taylor., 2011).The chemical structure of polysaccharide from alginate is similar to the bacterial cell wall, so, therefore, as this structure is recognized by host receptors, the immune response is then started by haemocyte proliferation.Amparyup et al. (2013) noted that Phenol Oxidase is the key role of innate immune in shrimp.PO contributes in melanogenesis by transforming phenols to quinones, and polymerizing to form melanin. Melanin assists in preventing pathogens from growing and reproducing.Other parameters on the rise of immune response were indicated by the increasing activity of two enzymes involved in Reactive Oxygen Species (ROS) scavenging namely Super Oxide Dismutase and Catalase to counter the production of radical species production caused by bacterial infection (Yudiati et al., 2019, Messina et al., 2014).ROS is beneficial to the immune system, but excessive amounts can be harmful (Cheng at al., 2004).As the immune is getting high, the generelated immune expression (Pro PhenolOxydase, β, 1-3 Glucan Binding Protein and Lectin) was also upregulated (Yudiati et al., 2019;Xing et al., 2020).As a result, the PL survival fed on Artemia enriched with 400 ppm alginate and L. bulgaricus probiotics reached the best one, in contrary compared to control.The synbiotics is in dose dependant manner.
Lactic acid bacteria (L.bulgaricus) as probiotics secreted exopolysaccharides (Daba et al., 2021).In their works Du et al. (2019) reported that Lactobacillus pentosus is able to increase disease resistance, gut bacterial diversity, and growth performance.Similar to this research, Roomiani et al. (2019) reported that application of L. bulgaricus in the diet of L. vannamei significantly boosted the immune parameters namely THC, PO, and respiratory burst activity (RBA).Healthy and balance gut microbiota could provide a blockade against pathogen invasion, start promoting host nutrient absorption through extracellular product secretion, and accelerate immune response (Libertucci & Young., 2019).Garces et al., (2015) noted that L. pentosus H 15 outcompeted the pathogenic V. alginolyticus.The lactic acid bacteria is selectively stick on to mucosal surfaces, create cell-bound biosurfactants, and dislocating pathogenic strains.
Figure 2 shows that L. vannamei PL fed on Artemia enriched with probiotics, alginate and/or FNCC-004 probiotics are similar in terms of weight gain.
This data suggested that the innate immune which boost by alginate is characterised by fast response immune and reach the peak at 14 days (Yudiati et al., 2019;Cheng et al., 2005).Since this works was conducted in 14 days, the weight gain was not affected specifically.Though, all PL fed on enriched Artemia with alginate and probiotic treatments tend to be higher than control.Our study revealed that L. vannamei grow out shrimps fed on alginate supplementation resulted better growth than control (Yudiati et al., 2016).Unfortunately, so far, there is no specific information regarding to the growth of shrimp fed on synbiotics alginate and lactic acid bacteria.
Figure 3 shows L. vannamei PL survival rate fed on Artemia enriched with probiotics, alginate and/or FNCC-004 probiotics at 7 days observation after VpAHPND challenge.In general, all treatments are similar at early days after challenge and late days after challenge.It is interesting to highlight, at 2,3, and 4 days after immersion  V. parahaemolyticus was identified as the causative agent of acute hepatopancreatic necrosis which caused 100% mortality first 35 days of the post larva stage caused by severe hepatopancreas atrophy (Choi et al., 2017).The conventional techniques, such as antibiotics and disinfectants, have had limited success in preventing or curing AHPND.Furthermore, their use has been linked to changes in the host gut microbiota and immunity, as well as the development of antibiotic resistance in bacterial pathogens.It was reported that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio harveyi isolated from L. vannamei pond and V. vulnificus were resistance to some beta lactam (Ampicilin, Amoxicilin dan Co-Amoxiclav) antibiotics (Yano et al., 2011).
In this present research, the PL resistance to severe VpAHPND was reached from L. vannamei PL fed on Artemie and probiotics enrichment.Al-Nabulsi et al. (2022) noted that they discovered the bactericidal effect from novel exopolysaccharide from Lactobacillus bulgaricus, and Streptococcus thermophilus.
Inspite of secreted exopolysaccharides, increase disease resistance and gut bacterial diversity, the presence of probiotics apparently improved midgut characteristics by enhancing microvilli and intestinal wall thickness and improved resistance to VpAHPND (Kewcharoen & Srisapoome, 2019).

Figure 1 .
Figure 1.Survival rate of post larva L. vannamei at 14 days of rearing fed on different Artemia enrichment.Different letter denotes significantly different at p<0.05.

Figure 2 .Figure 3 .
Figure 2. Weight gain of post larva L. vannamei at 14 days of rearing fed on different Artemia enrichment.Different letter denotes significantly different at p<0.05

Figure 4
Figure 4 depicted that L. vannamei PL fed on Artemia enriched with alginate 600 ppm and FNCC-004 probiotics treatment reached the highest survival rate at at 30; 40; and 50 minutes exposure (100; 96.67±5.77;83.33±5.77%),respectively.The lowest survival rate is reached in PL fed on Artemia enriched with probiotics, solely at 40; 50; and 60 minutes exposure (70±18.03;63±7.64;43±5.77%) after stress salinity.In this present study, the difference of osmotic stress is extremely high, and the PL was administered the salinity shock from 25 ppt to 0 ppt.Based on Yudiati et al. (2020) and Sudaryono et al. (2018), the osmotic stress on marine cultivan is correlated to the immune system.In this present research, in relevance to the survival rate data (Figure1), the increment of immune response occurs from L vannamei PL fed on Artemia-enriched with synbiotics of alginate and FNCC-004 probiotics.The certain dose of synbiotics resulted the best survival rate in acute osmotic stress due to salinity shock.

Figure 4 .
Figure 4. Survival rate of L. vannamei PL fed on different Artemia enrichment after 60 minutes stress salinity Different letter denotes significantly different at p<0.05